How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Axons are guided by both attractive and repulsive cues, which are members of evolutionarily conserved protein families. We are interested in the signaling mechanisms that function during attractive and repulsive axon guidance. The midline of the Drosophila embryonic CNS provides an ideal system to address these questions. Like its structural analog, the vertebrate floor plate, the fly midline is an intermediate target for many classes of navigating axons, which must decide whether or not to cross the midline. In the Drosophila CNS, the conserved guidance cue, Slit, functions to prevent axons from abnormally crossing the midline. Slit repulsion is mediated by the conserved family of Roundabout (Robo) receptors. The major aims of this application are: 1) to delimit and characterize the regions of the Robo receptor's cytoplasmic domain that are necessary and sufficient for Robo-mediated axon repulsion in response to the Slit ligand, 2) to assess the potential role of the SH3-SH2 adaptor protein Dreadlocks (Dock) and associated proteins in contributing to Robo repulsion, 3) to identify additional components involved in Slit and Robo signaling using a Drosophila genetic screen. A well established Drosophila transgenic approach will be used to determine which regions of Robo's cytoplasmic domain are required for repulsion. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, yeast two hybrid and co-immunoprecipitation will be used to investigate the potential role of Dock and associated proteins in Robo repulsion. In addition, established cell culture techniques will be used to address whether Slit stimulation of the Robo receptor regulates Dock/Robo interactions. To identify additional molecules involved in Robo function, a sensitized genetic screen will be performed. The proposed genetic screening strategy has already been successfully used on a small scale to identify genes that may play important roles during Robo repulsion. Deciphering the mechanisms that mediate Robo repulsive axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for nerve regeneration.